Figure 1.
Cells isolated from canine cartilage and synovium were cultured in 2D with (primed, -P) or without (unprimed, -U) a growth factor cocktail (see Materials and Methods). Surface marker expression by flow cytometry and label-free proteomic profiling were assessed in chondrocytes and SDSCs at passage 2 (P2). Cells were subsequently evaluated for chondrogenic capacity in 3D pellet culture at passage 2 (P2).
Figure 2.
Morphological differences between unprimed and primed cells.
Phase contrast micrographs (20×) of canine chondrocytes and SDSCs at passage 4 cultured unprimed (left side) or primed (right side). Scale bar = 50 µm. Initial plating density was 12.5-fold greater for chondrocytes than SDSCs.
Figure 3.
Doubling times of canine chondrocytes and SDSCs.
Doubling times of canine (A) chondrocytes (unprimed (C-U), primed (C-P)) and (B) SDSCs (unprimed (S-U), primed (S-P)) from P1 to P4 (n = 5). Statistically significant differences relative to unprimed cells are represented as **p<0.01 and ***p<0.001.
Table 1.
Phenotype of canine chondrocytes (C-U, C-P) and SDSCs (S-U, S-P) at passage 2.
Figure 4.
Examples of differential protein expression in canine chondrocytes and SDSCs.
A) Example of differential expression of collagen alpha-1(II) chain. The plot represents one isotopic signal from the mass spectrum of peptide GFTGLQGLPGPPGPSGDQGASGPAGPSGPR at 1337.1 m/z and 45.6 min retention time. Unprimed chondrocytes (C-U) cells exhibit strong accumulation of this protein, compared to primed chondrocytes (C-P) samples or either S-U or S-P samples B) Example of differential expression of aminopeptidase N (CD13). Plot represents one isotopic signal from the mass spectrum of peptide ESALLYDPQSSSIGNK at 854.9 m/z and 46.3 min retention time. Primed SDSCs (S-P cells) exhibit strong accumulation of this protein in response to growth factor priming; in comparison, neither C-P nor unprimed SDSCs (S-U cells) show a significant signal.
Table 2.
Most prominent differentially expressed proteins found in the proteomics analysis in chondrocytes.
Table 3.
Most prominent differentially expressed proteins found in the proteomics analysis in chondrocytes and SDSCs.
Table 4.
Most prominent differentially expressed proteins found in the proteomics analysis in SDSCs.
Figure 5.
Biochemical properties of canine chondrocyte- and SDSC-pellet culture.
(A, C) Glycosaminoglycan (GAG) content (g) normalized to DNA (g). After 28 days in culture, primed cells produced significantly more GAG/DNA. (B, D) Collagen content (g) normalized to DNA (g). Primed SDSCs (S-P cells) produced more collagen after 14 or 28 days. Results are shown as mean ± SD (n = 5). Statistically significant differences relative to unprimed cells are represented as *p<0.05, **p<0.01 and ***p<0.001.